Jacob Notbohm

Unjamming and cell shape in the asthmatic airway epithelium

From coffee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems-both inert and living-have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. The unjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phases remain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked to cell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scaling predicts a counter-intuitive relationship between jamming, cell shape and cell-cell adhesive stresses that is borne out by direct experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigation of bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelial dynamics play a prominent role.

Method for Characterizing Nanoscale Wear of Atomic Force Microscope Tips

Atomic force microscopy (AFM) is a powerful tool for studying tribology (adhesion, friction, and lubrication) at the nanoscale and is emerging as a critical tool for nanomanufacturing. However, nanoscale wear is a key limitation of conventional AFM probes that are made of silicon and silicon nitride (SiNx). Here we present a method for systematically quantifying tip wear, which consists of sequential contact-mode AFM scans on ultrananocrystalline diamond surfaces with intermittent measurements of the tip properties using blind reconstruction, adhesion force measurements, and transmission electron microscopy (TEM). We demonstrate direct measurement of volume loss over the wear test and agreement between blind reconstruction and TEM imaging. The geometries of various types of tips were monitored over a scanning distance of approximately 100 mm. The results show multiple failure mechanisms for different materials, including nanoscale fracture of a monolithic Si tip upon initial engagement with the surface, film failure of a SiNx-coated Si tip, and gradual, progressive wear of monolithic SiNx tips consistent with atom-by-atom attrition. Overall, the method provides a quantitative and systematic process for examining tip degradation and nanoscale wear, and the experimental results illustrate the multiple mechanisms that may lead to tip failure.

Preventing Nanoscale Wear of Atomic Force Microscopy Tips Through the Use of Monolithic Ultrananocrystalline Diamond Probes

Nanoscale wear is a key limitation of conventional atomic force microscopy (AFM) probes that results in decreased resolution, accuracy, and reproducibility in probe-based imaging, writing, measurement, and nanomanufacturing applications. Diamond is potentially an ideal probe material due to its unrivaled hardness and stiffness, its low friction and wear, and its chemical inertness. However, the manufacture of monolithic diamond probes with consistently shaped small-radius tips has not been previously achieved. The first wafer-level fabrication of monolithic ultrananocrystalline diamond (UNCD) probes with <5-nm grain sizes and smooth tips with radii of 30-40 nm is reported, which are obtained through a combination of microfabrication and hot-filament chemical vapor deposition. Their nanoscale wear resistance under contact-mode scanning conditions is compared with that of conventional silicon nitride (SiN(x)) probes of similar geometry at two different relative humidity levels (approximately 15 and approximately 70%). While SiN(x) probes exhibit significant wear that further increases with humidity, UNCD probes show little measurable wear. The only significant degradation of the UNCD probes observed in one case is associated with removal of the initial seed layer of the UNCD film. The results show the potential of a new material for AFM probes and demonstrate a systematic approach to studying wear at the nanoscale.

Cellular Contraction and Polarization Drive Collective Cellular Motion

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity.

Contractile forces regulate cell division in three-dimensional environments

Live-cell imaging, combined with mapping of 3D matrix displacements, identifies sites at which cells apply contractile forces to the matrix and reveals roles for physical forces in cell division.

Microbuckling of fibrin provides a mechanism for cell mechanosensing

Biological cells sense and respond to mechanical forces, but how such a mechanosensing process takes place in a nonlinear inhomogeneous fibrous matrix remains unknown. We show that cells in a fibrous matrix induce deformation fields that propagate over a longer range than predicted by linear elasticity. Synthetic, linear elastic hydrogels used in many mechanotransduction studies fail to capture this effect. We develop a nonlinear microstructural finite-element model for a fibre network to simulate localized deformations induced by cells. The model captures measured cell-induced matrix displacements from experiments and identifies an important mechanism for long-range cell mechanosensing: loss of compression stiffness owing to microbuckling of individual fibres. We show evidence that cells sense each other through the formation of localized intercellular bands of tensile deformations caused by this mechanism.

A model for compression-weakening materials and the elastic fields due to contractile cells

We construct a homogeneous, nonlinear elastic constitutive law that models aspects of the mechanical behavior of inhomogeneous fibrin networks. Fibers in such networks buckle when in compression. We model this as a loss of stiffness in compression in the stress–strain relations of the homogeneous constitutive model. Problems that model a contracting biological cell in a finite matrix are solved. It is found that matrix displacements and stresses induced by cell contraction decay slower (with distance from the cell) in a compression weakening material than linear elasticity would predict. This points toward a mechanism for long-range cell mechanosensing. In contrast, an expanding cell would induce displacements that decay faster than in a linear elastic matrix.

Three-Dimensional Analysis of the Effect of Epidermal Growth Factor on Cell-Cell Adhesion in Epithelial Cell Clusters

The effect that growth factors such as epidermal growth factor (EGF) have on cell-cell adhesion is of interest in the study of cellular processes such as epithelial-mesenchymal transition. Because cell-cell adhesions cannot be measured directly, we use three-dimensional traction force microscopy to measure the tractions applied by clusters of MCF-10A cells to a compliant substrate beneath them before and after stimulating the cells with EGF. To better interpret the results, a finite element model, which simulates a cluster of individual cells adhered to one another and to the substrate with linear springs, is developed to better understand the mechanical interaction between the cells in the experiments. The experiments and simulations show that the cluster of cells acts collectively as a single unit, indicating that cell-cell adhesion remains strong before and after stimulation with EGF. In addition, the experiments and model emphasize the importance of three-dimensional measurements and analysis in these experiments.

A cytoskeletal clutch mediates cellular force transmission in a soft, three-dimensional extracellular matrix

Quantitative analysis of the pairwise dynamics of the actin cytoskeleton, focal adhesions, and ECM fibrils reveals how cytoskeletal dynamics drive matrix deformation and cell motility for primary human fibroblasts embedded in a 3D fibrin matrix.

Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer

Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer.